This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ligand K-edge XAS is a direct probe of ligand metal bonding. We have developed this methodology to investigate the electronic structures of model complexes and protein active-sites of Cu-S and Fe-S clusters. Our previous results showed that changing a [unreadable]2-S-sulfide bridge of a localized reduced Fe2S2 cluster to a [unreadable]-3-S-sulfide bridge reduces the anti-ferromagnetic coupling interaction leading to delocalized ground states in Fe3S4 and Fe4S4 clusters. We have found that there is a significant reduction of Fe-S bond covalency in all the protein active-sites relative to the models, which can be attributed to H-bonding in the protein. Particularly, the covalency of the tetranuclear [Fe4S4] cluster in HiPIP was very different than that in ferredoxin. The method has been extended to define the non-innocent nature of the dithiolene ligands in determining the redox properties of a classic series of Ni-dithiolene complexes. We plan to evaluate the generality of the difference between HiPIPs and ferredoxins and systematically study the effect of H-bonding, solvent interaction and effect of changing dielectric field around these clusters using well-characterized model complexes and proteins. We will use current results on [Fe3S4] clusters to understand the electronic structures of [MFe3S4] complexes which are models for the active sites of heteronuclear clusters including nitrogenase, CODH, etc. The effects of Cys->Ser mutation on the electronic structures of mononuclear, binuclear and tetranuclear clusters will be evaluated. The non-heme iron active sites of superoxide reductase and nitrile hydratase will be studied using S K-edge to understand the oxidation level of the Fe-S bonds present in the active site and their contribution to tuning reactivity. We will also explore P450 type active sites and the effect of substrate binding on the Fe-S bond.